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EWOD-driven droplet microfluidic device integrated with optoelectronictweezers as an automated platform for cellular isolation and analysis

Gaurav J. Shah,*a Aaron T. Ohta,bc Eric P.-Y. Chiou,a Ming C. Wuc and Chang-Jin CJ Kima

Received 2nd December 2008, Accepted 4th March 2009

First published as an Advance Article on the web 16th March 2009

DOI: 10.1039/b821508a

We report the integration of two technologies: droplet microfluidics using electrowetting-on-dielectric

(EWOD) and individual particle manipulation using optoelectronic tweezers (OET)in one

microfluidic device. The integrated device successfully demonstrates a sequence involving both EWOD

and OET operations. We encountered various challenges during integration of the two different

technologies and present how they are addressed. To show the applicability of the device in cellular

biology, live HeLa cells are used in the experiments. The unique advantages of EWOD and OET make

their integration a significant step towards a powerful tool for many applications, such as single cell

studies involving multiplexed environmental stimuli.

Background and motivation

Manipulation of individual particles in solution

Theability to manipulate specific cells individually and isolate them

is attractive for cellular biology, facilitating, for instance, the study

of single cell behavior and cell-to-cell interactions. Microfluidic

systems with an ability to control cells and cellular microenviron-

ments have been playing an increasingly important role in such

studies.1,2 Various mechanisms have been employed to manipulate

cells, e.g. magnetic,3,4 optical,5,6 acoustic,7 electrical8 (e.g. electro-

phoretic,9 dielectrophoretic (DEP)10,11) and hydrodynamic.12

However, most of the manipulation techniques mentioned above

cannot achieve high resolution, high throughput and low power-

consumption at the same time. For instance, although optical

tweezers13 are a powerful technology for trapping cellular and

organic particles, particularly for single particle manipulation, they

have limitations such as the tendency for photodamage and the

trade-off between resolution and operational area.14 Dielec-

trophoresis-basedtechniques,15,16,8on theother hand, typicallyhave

large operational area but suffer from limited flexibility of manip-

ulation due to fixed electrode patterns, making it difficult to

manipulate a specific cell from within a population.

Optoelectronic tweezers (OET) are an exciting technique that

combines many of theadvantages of optical tweezers andDEP.17By

opticallycontrolling the electric fielddistribution in the device, OET

uses optical intensities of only 0.01 to 1 W cm2, drastically reduced

from that of optical tweezers, allowing the use of a computer

projector or LEDs.In addition, lenses with lownumerical apertures

(NA) can be used, thus increasing the manipulation area. Thus,

OET retains the flexibility and dynamic control of optical trapping,

making individual manipulation of specific particles using real-time

feedback possible over a relatively large area, based on not just

physical or electrical, but also optical properties.18,19

Cell studies in droplet microfluidics

In addition to individual cell manipulation, single cell studies

often require subjecting individual cells to different environ-

mental conditions and stimuli, such as a range of concentrations

of nutrients or drugs, or varying mechanical or electrical signals.

Although isolation of individual cells has been achieved with

continuous microfluidics, no viable approach has been proposedto independently control the environmental conditions of each

isolated cell in the same device. While many reports involved the

study of cellular response to single or a small number of

stimuli,20,21 there have been just a few attempts to conduct mul-

tiplexed assays,22,23 since the latter involves a complex network of

pumps, microvalves, latches and interconnected channels.24 On

the other hand, isolation of environments for individual cells is

rather natural in the case of droplet microfluidics,25 such as that

driven by electrowetting-on-dielectric (EWOD),2628 since each

isolated droplet can be supplied with independent multiplexed

stimuli. Aqueous droplets in air or oil can be actuated by elec-

trowetting,29 DEP,30,31 or a combination of the two. By appro-

priately choosing the actuation sequence, precise andindependent control of multiple droplets can be achieved while

minimizing cross-contamination.

Target separation is a key capability desired to make EWOD

a powerful lab-on-a-chip platform. After isolating the target

particles from non-target particles within a droplet by the

differences in their properties (e.g. electrophoretic,32 DEP,33 and

magnetic34,35), one can split the droplet into a target-concen-

trated and a target-depleted daughter droplet by EWOD actua-

tion to the mother droplet. While some biochemical applications

of EWOD have recently been shown,3638 actuation of droplets

containing live cells have not been dealt with until very recently.

aUCLA, 420 Westwood Plaza, Engineering IV Bldg, Los Angeles, CA,USA. E-mail: [email protected]; [email protected]; [email protected];Fax: +1-310-206-2302; Tel: +1-310-825-3977bUniversity of Hawaii, 2540 Dole Street, Holmes Hall 483, Honolulu, HI,USA. E-mail: [email protected]; Fax: +1-808-956-3427; Tel: +1-808-956-8196cUC Berkeley, 261M Cory Hall #1770, Berkeley, CA, USA. E-mail: [email protected]; Fax: +1-510-666-2502; Tel: +1-510-643-0808

Electronic Supplementary Information (ESI) available: (1) Discussionof challenges faced by first generation integrated device; (2) schematicof the experimental setup; (3) video of the sequence of EWOD andOET operation on the integrated device. See DOI: 10.1039/b821508a/

1732 | Lab Chip, 2009, 9, 17321739 This journal is The Royal Society of Chemistry 2009

PAPER www.rsc.org/loc | Lab on a Chip


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For example, DEP force was used to concentrate cells39,40 on an

EWOD device immersed in oil. The addition of a small amount

of surfactants, such as Pluronic F68, prevents device surface

fouling by cells without the use of oil41 with minimal effects on

cell vitality. Magnetic separation of cells labeled with magnetic

beads on an EWOD device in an air environment has also been

demonstrated using the surfactant approach of minimizing

surface fouling.42

Integrating EWOD and OET

As discussed above, so far the cell manipulation techniques to

manipulate individual particles within a population could not be

exercised under the EWOD-based droplet microfluidic platform.

On the other hand, while OET may allow individual particle

manipulation under the usual continuous microfluidics, isolating

them into discrete environments is challenging. A device that

combines the unique capabilities of OET cell manipulation ( i.e.,

specific, dynamic, individual manipulation over a relatively large

area) and EWOD microfluidics (i.e., generation and manipula-

tion of discrete droplets on chip by only electric signals) would be

a valuable tool for research in cellular biology.

For example, the droplets can act as microchambersproviding controlled microenvironments for the independent

and multi-parameter study of the isolated cell(s). Reagents

containing various concentrations of drugs/nutrients/salts can

be introduced to each of the droplets independently. Old/waste

medium can be removed and fresh medium replenished using

EWOD operations of cutting and merging, as envisioned in

Fig. 1. Moreover, specific cell(s) can be picked from the rest of

the population based on differences in optical or dielectric

properties using OET, and isolated into a separate droplet using

EWOD. Integration of EWOD and OET has first been intro-

duced in a preliminary report43 demonstrating both the opera-

tions on the same device, albeit separately. However, a complete

sequence of the combined EWOD-OET operations was notpossible due to the mutual exclusivity of EWOD and OET

operational areas. In this work, we overcome the limitations by

developing new device designs and incorporating a modified

OET mechanism. A complete, uninterrupted sequence consist-

ing of successive operations of EWOD, OET, and then EWOD

again, demonstrates the success.

Device design

Both EWOD and OET are now relatively well-established tech-

nologies, having been independently used for a variety of appli-

cations.

44,45

For the details of principle and theory of each ofthese technologies, the reader is referred to earlier reports.27,14 In

this paper, we focus on the design and fabrication of the inte-

grated EWOD-OET device. We present the challenges encoun-

tered in prior attempts (first-generation device43) and how the

new device overcomes them.

First-generation integrated device

We first briefly review the first-generation integrated device43 in

order to highlight the challenges it faced. As seen in Fig. 2(a), the

device consisted of two chips (referred to as EWOD chip and

OET chip according to what actuation voltage is supplied to

them), held apart by a spacer. A standard OET device was usedfor the OET chip.41 The EWOD chip was designed similar to the

standard EWOD device27 except for some modifications needed

to incorporate OET. One of the electrodes (OET electrode)

was dedicated to act as the reference electrode for the OET

voltage, defining the OET region (Fig. 2(a)).43 To ensure that

the OET-driving electric field was applied across the droplet in

the illuminated state, the dielectric layer over the OET electrode

was removed. Droplet manipulation into and out of the dielec-

tric-free OET region was performed using surrounding EWOD

electrodes, where OET could not be operated due to the presence

of the dielectric.

Fig. 1 An envisioned application of the integrated device in cellular

biology, where each droplet can present an isolated microenvironment to

the cell(s) it contains. (1a) Drugs, nutrients and/or salts can be introduced

in the form of droplets independently to each microenvironment. (1b)

Cells are collected by parallel LOET manipulation using a dynamic

optical pattern generated using real-time image feedback. (1c1d) Waste/

old medium can be removed by droplet splitting using EWOD. (2a3a)

The steps (ad) can be repeated to introduce other drugs, nutrients etc. or

combinationsthereof, andto remove the oldmedium in theform of waste

droplets while retaining the cells.

This journal is The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 17321739 | 1733


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Challenges faced by the first-generation device

Although the steps of both EWOD and OET were individually

demonstrated on the first-generation device,43 the mutually exclu-

sive nature of their operational areas led to difficulties in demon-

strating the complete sequence of EWOD-OET operations.

Specifically, the lack of OET in the EWOD region restricts particle

manipulation to therelatively smallOET regiononly,whilethe lack

of EWOD over the OET region entails difficulties for sustaining the

particle distribution against the viscous drag force from the fluidic

movement in the surrounding liquid. The viscous drag can be esti-

mated using Stokes equation, which indicates that the viscous drag

force on a particle inside a viscous fluid is proportional to the fluid

velocity.It wasfoundthatthe inherently stronger flowalongthe free

liquidair interfacein droplet microfluidics46

transports the particlesthat are swept into suspension by the receding meniscus, preventing

them from being introduced into the OET region. These limitations

are described in more detail in the ESI.

Recognizing that this approach has difficulties arising from the

mutual exclusiveness of theEWOD and OET regions, we propose

the second-generation device, which incorporates a lateral-field

OET (LOET) chip14,47 with an unmodified EWOD chip.

Second-generation device: EWOD-LOET

Fig. 2(b) illustrates the second-generation integrated device. The

issues identified above are resolved by replacing the standard

OET chip with an LOET chip, which is essentially a coplanar

OET device, i.e. both the electrodes for OET operation are on thesame substrate. Unlike the standard OET device, two arrays of

interdigitated electrodes are patternedin themetallayerunderthe

photoconductive layer of the LOET device.47 The OET driving

voltage is applied between the two arrays of interdigitated elec-

trodes. In the dark state, most of the electric field drops across the

photoconductive amorphous silicon (a-Si) layer instead of the

droplet. When illuminated, the resistivity of the photoconductive

layer drops so as to produce a lateral electric field gradient in the

droplet between the interdigitated electrodes. The modulation of

the electric field distribution within the liquid is therefore still

driven by the optical pattern. An advantage of LOET over the

standard two-substrate OET is that the single sided LOET can be

used with a wide variety of opposing surfaces facilitating itsintegration with other microdevices.14 LOET thus eliminates the

issues faced in the first-generation device. Since both the high and

ground for the OET voltage are on the OET chip itself, the other

(i.e. EWOD) chip no longer needs to accommodate OET in it (i.e.

no patterning of the dielectric or hydrophobic layers is required).

This new approach not onlyeliminates the OET(only) region of

the first-generation device but also expands the OET capability

into the previously EWOD (only) regions. Since the EWOD

and OET regions are no longer mutually exclusive, the problems

described for the first-generation device are no longer encoun-

tered in the second-generation device.

Fig. 2 Comparison of the first- and second-generation EWOD-OET integrated device (not to scale). (a) The first generation43 combines EWOD and

OET, as shown in cross-section (left) and top view (right). The OET chip has unpatterned ITO, photoconductive and hydrophobic layers. The EWOD

chip has electrodes defined in the ITO layer, coated with dielectric and hydrophobic layers. The dielectric layer in the EWOD chip is patterned in the

OET region so as to allow the voltage drop in the droplet required for OET particle manipulation. OET is non-operational in regions where the

dielectric layer is present, i.e. the EWOD region, where EWOD microfluidics can be performed. In general, a hydrophilic opening is made over none,

some or all the OET region. (b) The second generation combines EWOD and new LOET, as shown in cross-section (left) and top view (right). The

interdigitated LOET electrodes define the LOET actuation area, overlapping the EWOD actuation electrodes. Unlike the first-generation design, LOET

actuation does not require any modification on the EWOD chip; hence, there are no mutually exclusive EWOD or OET regions. (EWOD lead wires not

shown for clarity).



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Materials and methods

Device fabrication

Both EWOD and LOET chips were fabricated using standard IC

microfabrication processes. To fabricate the EWOD chip,

EWOD electrodes (1 mm2) were defined of an indium-tin oxide

(ITO) (1400 A) layer on a 700 mm-thick glass substrate (Tech-

Gophers Inc.). Cr/Au ($100/1000 A) was deposited and

patterned to define the contact pads and electrode labels for

easier visualization. Next, a silicon nitride layer ($1 mm) was

deposited using plasma-enhanced chemical vapor deposition

(PECVD) and patterned to define the dielectric layer. A Cytop

(Asahi Inc.) layer ($1 mm) was spin-coated on top and annealed

at 200 C to make the surface hydrophobic.

The LOET chip was fabricated on a silicon wafer. After

thermal oxidation to form a silicon dioxide layer (1 mm) for

electrical isolation, the LOET electrodes are created by electron-

beam evaporation of aluminium using a lift-off process. A

hydrogenated amorphous silicon (a-Si:H) layer (0.8 mm) is

deposited over the aluminium electrodes using PECVD. The a-Si

over the electrical bias pads is removed by reactive-ion etching. A

thin layer of Teflon AF was spin-coated on the a-Si and

annealed at 150 C, rendering the surface hydrophobic.

Since the OET force is strongest near the LOET chip and the

particles/cells settledue to gravity, theLOET chip was placedon the

bottom with the EWOD chip on top. Double-sided adhesive tape

(thickness ~100 mm) was used as the spacer between the two chips.

Experimental setup

The integrated EWOD-LOET device was mounted on the optical

setup used for OET operations (schematic of the setup is shown

in the ESI). The output of a computer projector (Dell 2400MP)

was collimated and focused onto the LOET chip through a 10

objective lens. This lens also serves as the observation objective.

A fiber illumination is used to provide the background illumi-

nation necessary to view the microscale particles, and a CCD

camera is used to capture the microscope images.

The EWOD and LOET devices were connected to their

respective voltage supplies. A driving voltage of 35 Vrms at 200

kHz was used for LOET actuation. A driving voltage of $120

Vrms at 20 kHz was used for EWOD actuation, although most of

the applied potential is expected to fall across the dielectric layer

and does not affect the interior of the droplet. During the OET

manipulation, one of the electrode arrays was connected to the

LOET voltage supply, and the other was grounded. During the

EWOD fluidic operation, the LOET electrodes act as the ground.

Although only one array of LOET electrodes can provide the

grounding, EWOD actuation was found to be more effective

when both of the LOET arrays were grounded, particularly

during droplet cutting.

Reagents used

In order to show the applicability of the integrated EWOD-OET

device for biological applications, live HeLa cells suspended in an

isotonic buffer containing 8.5% sucrose and 0.3% dextrose in DI

water (s 5 m S m1) were chosen for the experiment. As

described in ref. 14, the complex permittivity of the live cells with

respect to the medium result in positive OET for the frequency

of the electric field used. The positive OET means that the

particles are attracted to the electric field maxima, i.e. regions

illuminated by the optical pattern. The hydrophobic surface

required for EWOD actuation is highly prone to fouling from the

cells. The addition of small quantities of surfactants alleviates the

fouling to allow actuation of cell samples on EWOD.41,42

Therefore, $0.2% Pluronic surfactant F68 (Sigma-Aldrich) was

added to the solution.

Results and discussion

Experimental results

We demonstrate the sequential operations of EWOD-driven

droplet microfluidics and LOET-driven optically controlled cell

manipulation on the same device, without any mutually exclusive

regions of operation on our integrated device. Fig. 3 shows the

schematic of the sequence of the sample EWOD-OET operations

performed. As discussed earlier (Device design), it is important

to ensure that the cell separation or distribution generated by

OET is not disrupted by the subsequent microfluidic movements.To prevent the disruption, droplet positioning before the LOET

steps, location of the collection region during the LOET steps,

and the splitting sequence after the LOET operations must be

chosen appropriately.

The droplet position prior to LOET manipulation was

experimentally determined, so as to minimize the collected cells

from being re-distributed by the droplet movement during the

subsequent cut. The droplet is first positioned accordingly using

EWOD (Fig. 3(ab)). Next, LOET operations are performed on

the particles using a dynamic optical pattern, in this case to move

the particles from the right (depleted) to the left (collected)

regions of the droplet (Fig. 3(bc)). Multiple sweeps may be

performed to cover the entire droplet if the LOET operationalarea is small relative to the droplet size (Fig. 3(cd)). After the

LOET operations are completed, the droplet is split using

EWOD (Fig. 3(e)) into the collected droplet containing most

of the target cells and the depleted droplet depleted of the cells

(Fig. 3(f)).

Fig. 4 and 5 show image sequences from the actual experiment.

Images for the LOET particle manipulation (Fig. 5) were

recorded using the CCD camera, which is part of the optical

setup. Since the field of view through the 10 objective used for

LOET ($0.5 mm 0.4 mm) was not large enough to capture the

entire droplet movements by EWOD, a lower magnification

video camera was used (Fig. 4).

A droplet ($350 nL) containing live HeLa cells ($6 104

mL1) is placed on the device (Fig. 4(a)) sandwiched between the

two chips. As discussed above, the droplet is stretched by EWOD

so as to position it for the subsequent steps (Fig. 4(b)). Next, cell

manipulations are performed using LOET, viz. cells are collected

leftwards by sweeping a computer-generated optical pattern

from right to left across the field of view (Fig. 4(bc)). The bright

spot in Fig. 4(bd) shows the entire illuminated area; the optical

pattern within the illuminated area is seen more clearly in Fig. 5.

Whereas a manually controlled rectangular bar pattern is used in

this simplified demonstration to collect and move cells across the

droplet, more complex dynamic optical patterns can be readily



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generated (e.g. based on real-time image-feedback) to perform

much more intricate cell manipulations.19,43

In Fig. 5, the EWOD electrode patterns, outlined by white

broken lines, can be used as reference to see the movement of

cells by LOET. Since the cells are acted upon by positive DEP,

they get attracted to the illuminated optical pattern (Fig. 5(a)).

As the pattern moves to the left (indicated by the dark (red)

arrow), the cells it crosses get collected and move along with the

pattern (Fig. 5(bc)). The image collects more cells as it

moves leftwards across electrodes to get to the collection area

(Fig. 5(df)). As the LOET illuminated area is smaller than the

electrode dimensions, multiple sweeps across the droplet are

performed, each time collecting the cells in the collected region

(Fig. 4(cd)), at a location away from the droplet edge to mini-

mize disturbance due to the stronger flow along the free liquid-air

interface. After the LOET operations are completed, the droplet

is split by EWOD, further stretching it in both directions

(Fig. 4(e)) into the collected (left) and depleted (right) daughter

droplets (Fig. 4(f)).

In the present experiment, $19 live cells were present in theoriginal droplet, out of which about 13 were collected into the

collected droplet and 6 were left in the depleted droplet. While

most cells move for a short distance (0.51 mm), they tend to get

stuck over the relatively long distance (up to 23 mm) of

manipulation required due to the large electrode dimensions in

the current device. Dirt particles present in the path can also

block the cell movement.

Discussion of results

A combined sequence of EWOD and OET operations for cell

handling as shown in this sample demonstration is a significant

development towards a powerful integrated device for cellstudies. By eliminating mutually exclusive regions for OET and

EWOD by using LOET, we have solved the fundamental chal-

lenges in combining the two techniques and their unique benefits.

All experiments in this report were performed with air as the

surrounding medium. When the device is immersed in oil4850

rather than dry in air,29,51,27 a thin layer of oil present between the

hydrophobic device surface and the aqueous droplet52 greatly

reduces the resistance against droplet sliding, making most of the

basic EWOD operations easier. The thin oil layer also separates

the particles in the droplet from the device surface, preventing

their adhesion on the surface.52 In addition, the surrounding oil

helps reduce evaporation of the EWOD droplets, helping to

maintain their size and concentration. Despite the conveniences,there have been some concerns regarding the use of silicone oil,

particularly in biological applications such as cell studies:

a. The surrounding silicone oil may restrict the exchange of

gases between the droplets and the atmosphere, which may be

unfavorable for biological cells in the droplet. The concerns

include a build-up of carbon dioxide in the droplets.

b. If the application requires certain events to be performed

directly on the device surface, such as electrochemical sensors

patterned on the surface, the thin layer of oil could hinder the

performance.

c. Ensuring that the oil does not leak makes the packaging of

the device much more challenging.

The increased likelihood of evaporation for the air-environmentcase (compared to the oil-environment case) is a concern as it may

lead to a changein concentrationof nutrients andsalts,whichcould

adversely affect the cell viability. Droplet evaporation is minimized

by sealing the gapbetweenthe device chips usinga sealing tapeso as

to locally saturate the surrounding air with water vapor.53

Although not utilized in the present report, significant prevention

of evaporation during much longer experiments (a few hours, as

opposed to several minutes in our case) has been reported using

a humidifiedatmosphereto store devices between actuationsteps.41

Likewise, we plan to use a humidified chamber for future experi-

ments, particularly those involving longer periods of operation.

Fig. 3 Schematic representation of the sequential EWOD and OET

operations performed on the integrated device. The thick (blue) arrows

indicate droplet movement by EWOD, while the thin (red) arrows indi-

cate the movement of the OET optical pattern. (a) A droplet containing

cells is placed in the device. (b) To avoid disturbing the LOET-generated

particle distribution due to hydrodynamic forces during the subsequent

cut, the droplet is brought into a stretched position by EWOD prior to

LOET operations. The LOET optical pattern is then projected and

moved across the droplet to collect the cells on the left. (c) If the droplet

dimensions are larger than the LOET area of operation, this may be

repeatedover multiple regions to cover theentire droplet. (d) The cells are

collected in one (collected) region, and (ef) the droplet is subsequently

split using EWOD into the collected droplet and the depleted droplet.



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It is worth discussing the effect of EWOD and OET (DEP)

actuations, and the unusual buffer used in the current experi-ments, on cell vitality. As discussed in Background and moti-

vation, the low concentration of Pluronic F68 has been shown

to have little effect on cell viability.41 Additionally, because most

of the EWOD voltage drops across the relatively thick dielectric

and hydrophobic layers, the cells are expected to remain largely

unaffected from the EWOD actuation voltage.41

DEP force has been used to safely filter and retain cells while

maintaining viability in perfusion cultures.54 The use of positive

DEP for OET actuation limits the conductivity of the buffer that

can be used in the present experiments, leading to the choice of

the isotonic sucrose/dextrose buffer. Although less favorable for

cells than saline buffers and media, it has been reported that if the

isotonic buffer is replenished every hour or so, no significant lossof viability is observed even under DEP actuation.55 Similar

commercially available buffers (e.g. Cytoporation medium

CP-T from Cyto Pulse Sciences Inc.) meant for electroporation

of live cells further suggest the viability of cells in the isotonic

buffer, provided adequate recycling.

The limitations of the present work of integration and future

steps to overcome them are discussed below. The dimensions of

the EWOD electrodes in the integrated device were arbitrarily

chosen to be 1 mm 1 mm, since this is the typical geometry for

EWOD devices. Noting the droplet is placed over two electrodes,

the 1 mm electrodes entail that the cells must be manipulated by

LOET over relatively long distances (up to 23 mm). Even

though the integrated movement was successfully demonstratedfor many cells, the task was challenging, particularly for the

positive DEP case where cells are being attracted to the substrate,

increasing the likelihood of adhesion. Even though most cells

were able to traverse over 0.51 mm, some got stuck over larger

distances, making LOET ineffective in moving them. A simple

solution to the problem of long-distance LOET travel would be

to scale down the EWOD electrode dimensions, such that LOET

manipulation is over a shorter distance.

The LOET electrode fingers were 10 mm wide with a gap of

5 mm. As such, the cells ($8 mm diameter) could not be moved

in the direction perpendicular to the fingers in the current

device. This limitation is, however, not fundamental. Suffi-

ciently small LOET electrode geometry can ensure two-dimensional manipulation, as has been demonstrated by

choosing a line width and gap smaller than the particle size.14

Since particle manipulation is still driven by the optical

pattern, individual particle manipulation is achievable with the

LOET chip. Although mammalian cells have been used in the

present demonstration, the same technique can be extended to

other cell types such as bacteria, yeast, etc. On the other hand,

sub-micron particles such as viruses (typically $100 nm in all

dimensions) pose a much greater challenge due to the r3 scaling

of DEP force and the increased significance of Brownian

motion relative to it.56

Fig. 4 Images showing the sequential operation of EWOD and LOET on the integrated device. Droplet manipulations are performed by EWOD (ab)

before and (ef) after the (bd) cell manipulation by LOET. Broken white lines are drawn to indicate droplet shape. The thick (blue) arrows seen in (a)

and (e) indicate EWOD actuation while the thin (orange) arrows seen in (bd) indicate LOET actuation. (a) Droplet ($350 nL) containing HeLa cells is

placed on the integrated device. (b) The droplet is positioned such that the LOET-generated cell distribution is not disturbed during the subsequent

droplet splitting. Starting from the right of the stretched droplet, (c) the cells are moved from right to left by LOET. (d) The cell manipulation is

performedacross thelength andwidthof the droplet by movingthe LOET objective lens with respect to thedevice, so as to collect most of thecells in the

left-side of the droplet. (The bright spot from the illumination for LOET indicates its entire operational area. The computer-generated optical pattern

used for LOET manipulation within the area is shown in Fig. 5). (e) After collection, the droplet is being split by EWOD. (f) The droplet has been split

into the collected (left) and depleted (right) droplets.



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With suitable optimizations to improve the performance for

a given application, the integrated EWOD-LOET device can

become a valuable tool for the cellular biology.

Conclusion

We successfully demonstrated a sequence of EWOD and OET

operations for a HeLa cell sample on an integrated device. The

fundamental issues faced by previous attempts to make such

a device were discussed, and the new design employing lateral-

field OET (LOET) to overcome the challenges was described.

Both EWOD and OET have unique and valuable characteristics

that make the reported integration promising. In particular, the

parallel manipulation of specific cells using OET with a low

power over a relatively large area, combined with the electrically

reprogrammable microfluidic operations of independent droplets

using EWOD, would enable isolated cell studies involving vari-

ations of multiple environmental stimuli.

Acknowledgements

The authors owe Dr Jian Gong for his help with experimental

setup and Dr Prosenjit Sen for his insightful discussions. The

appreciation is extended to Mr Joe Zendejas and other staff at

the UCLA Nanoelectronics Research Facility for help during

microfabrication. The authors also thank the staff of the

Microfabrication Laboratory and the Tissue Culture Facility of

UC Berkeley. This work was supported by NASA through

Institute for Cell Mimetic for Space Exploration (CMISE).

References

1 H. Andersson and A. van den Berg, Sens. Actuators, B, 2003, 92, 315325.

2 P. Chen, X. J. Feng, W. Du and B. F. Liu, Front. Biosci., 2008, 13,24642483.

3 N. Pamme, Lab Chip, 2006, 6, 2438.4 M. A. M. Gijs, Microfluid. Nanofluid., 2004, 1, 2240.5 K. Dholakia, W. M. Lee, L. Paterson, M. P. MacDonald,

R. McDonald, I. Andreev, P. Mthunzi, C. T. A. Brown,R. F. Marchington and A. C. Riches, IEEE J. Sel. Top. QuantumElectron., 2007, 13, 16461654.

6 J. P. Desai, A. Pillarisetti and A. D. Brooks, Annu. Rev. Biomed. Eng.,2007, 9, 3553.

7 M. Evander, L. Johansson, T. Lilliehorn, J. Piskur, M. Lindvall,S. Johansson, M. Almqvist, T. Laurell and J. Nilsson, Anal. Chem.,2007, 79, 29842991.

8 J. Voldman, Annu. Rev. Biomed. Eng., 2006, 8, 425454.9 C. R. Cabrera and P. Yager, Electrophoresis, 2001, 22, 355362.

10 J. El-Ali, P. K. Sorger and K. F. Jensen, Nature, 2006, 442, 403411.11 M. P. Hughes, Electrophoresis, 2002, 23, 25692582.12 S. M. Kim, S. H. Lee and K. Y. Suh, Lab Chip, 2008, 8, 10151023.13 D. G. Grier, Nature, 2003, 424, 810816.14 A. T. Ohta, P. Y. Chiou, H. L. Phan, S. W. Sherwood, J. M. Yang,

A. N. K. Lau, H. Y. Hsu, A. Jamshidi and M. C. Wu, IEEE J. Sel.Top. Quantum Electron., 2007, 13, 235243.

15 J. Yang, Y. Huang, X. B. Wang, F. F. Becker and P. R. C. Gascoyne,Anal. Chem., 1999, 71, 911918.

16 D. S. Gray, J. L. Tan, J. Voldman and C. S. Chen, Biosens.Bioelectron., 2004, 19, 771780.

17 P. Y. Chiou, A. T. Ohta and M. C. Wu, Nature, 2005, 436, 370372.18 A. T. Ohta, P. Y. Chiou, T. H. Han, J. C. Liao, U. Bhardwaj,

E. R. B. McCabe, F. Q. Yu, R. Sun and M. C. Wu, J.Microelectromech. Syst., 2007, 16, 491499.

19 P. Y. Chiou, A. T. Ohta and M. C. Wu, in MOE IEEE/LEOS Conf.Optical MEMS and Their Applications, Oulu, Finland, 2005, pp. 8384.

20 A. R. Wheeler, W. R. Throndset, R. J. Whelan, A. M. Leach,R. N. Zare, Y. H. Liao, K. Farrell, I. D. Manger and A. Daridon,

Anal. Chem., 2003, 75, 35813586.21 N. L. Jeon, H. Baskaran, S. K. W. Dertinger, G. M. Whitesides,

L. Van De Water and M. Toner, Nat. Biotechnol., 2002, 20, 826830.22 T. Thorsen, S. J. Maerkl and S. R. Quake, Science, 2002, 298, 580

584.23 B. G. Chung, L. A. Flanagan, S. W. Rhee, P. H. Schwartz, A. P. Lee,

E. S. Monuki and N. L. Jeon, Lab Chip, 2005, 5, 401406.24 J. Melin and S. R. Quake, Annu. Rev. Biophys. Biomol. Struct., 2007,

36, 213231.25 S. Y. Teh, R. Lin, L. H. Hung and A. P. Lee, Lab Chip, 2008, 8, 198

220.26 S. Pennathur, C. D. Meinhart and H. T. Soh, Lab Chip, 2008, 8, 20

22.27 S. K. Cho, H. J. Moon and C.-J. Kim, J. Microelectromech. Syst.,

2003, 12, 7080.28 V. Srinivasan, V. K. Pamula and R. B. Fair, Lab Chip, 2004, 4, 310

315.

29 J. Lee, H. Moon, J. Fowler, T. Schoellhammer and C.-J. Kim, Sens.Actuators, A, 2002, 95, 259268.

30 T. B. Jones, Langmuir, 2002, 18, 44374443.31 T. B. Jones, K. L. Wang and D. J. Yao, Langmuir, 2004, 20, 2813

2818.32 S. K. Cho, Y. J. Zhao and C.-J. Kim, Lab Chip, 2007, 7, 490498.33 Y. J. Zhao, U. C. Yi and S. K. Cho, J. Microelectromech. Syst., 2007,

16, 14721481.34 G. J. Shah, E. Pierstorff, D. Ho and C.-J. Kim, in Proc. Int. Conf.

Solid-State Sensors, Actuators and Microsystems, Lyon, France,2007, pp. 707710.

35 Y. Z. Wang, Y. Zhao and S. K. Cho, J. Micromech. Microeng., 2007,17, 21482156.

36 H. J. Moon, A. R. Wheeler, R. L. Garrell, J. A. Loo and C.-J. Kim,Lab Chip, 2006, 6, 12131219.

Fig. 5 Image sequence (as seen through the LOET objective lens)

showing the cell manipulation by LOET and the optical pattern used for

it. Broken white lines indicate the EWOD electrode features. The dark

(red) arrows indicate the direction of movement of the LOET opticalpattern. (a) The pattern used here is a simple rectangle (manually

controlled by mouse). Since the cells experience positive OET at the

frequency used, they are attracted to the pattern. (b) As it moves to the

left, the pattern collects more cells. (c) The movement of the cells with

the pattern can be seen with respect to the EWOD electrode features and

the droplet meniscus. (df) The pattern sweeps across multiple EWOD

electrodes starting from the right of the droplet, collecting the cells in the

collected region at the left (See also Fig. 4(bd)).



8/8

37 R. B. Fair, A. Khlystov, T. D. Tailor, V. Ivanov, R. D. Evans,P. B. Griffin, V. Srinivasan, V. K. Pamula, M. G. Pollack andJ. Zhou, IEEE Des. Test Comput., 2007, 24, 1024.

38 D. Chatterjee, B. Hetayothin, A. R. Wheeler, D. J. King andR. L. Garrell, Lab Chip, 2006, 6, 199206.

39 P.-W. Huang, T.-T. Wang, S.-W. Lin, Y.-C. Chang and S.-K. Fan, inProc. 1st IEEE Int. Conf. Nano/Micro Engineered and MolecularSystems, Zhuhai, China, 2006, pp. 14181421.

40 S. K. Fan, P. W. Huang, T. T. Wang and Y. H. Peng, Lab Chip, 2008,8, 13251331.

41 I. Barbulovic-Nad, H. Yang, P. S. Park and A. R. Wheeler, Lab Chip,2008, 8, 519526.

42 G. J. Shah, J. L. Veale, Y. Korin, E. F. Reed, H. A. Gritschand C.-J. Kim, in Proc. Solid-State Sensors, Actuators andMicrosystems Workshop, Hilton Head Island, SC, USA, 2008, pp.2831.

43 G. J. Shah, P. Y. Chiou, J. Gong, A. T. Ohta, J. B. Chou, M. C. Wuand C.-J. Kim, in Proc. IEEE Int. Conf. MEMS, Istanbul, Turkey,2006, pp. 129132.

44 F. Mugele and J. C. Baret, J. Phys.: Condens. Matter, 2005, 17, R705R774.

45 K. Dholakia, Nat. Mater., 2005, 4, 579580.

46 H. W. Lu, F. Bottausci, J. D. Fowler, A. L. Bertozzi, C. Meinhart andC.-J. Kim, Lab Chip, 2008, 8, 456461.

47 A. T. Ohta, S. L. Neale, H. Y. Hsu, J. K. Valley and M. C. Wu, in Int.Conf. on Optical MEMS and Nanophotonics, Freiburg, Germany,2008, pp. 78.

48 M. G. Pollack, R. B. Fair and A. D. Shenderov, Appl. Phys. Lett.,2000, 77, 17251726.

49 Y. Fouillet and J. L. Achard, C. R. Phys., 2004, 5, 577588.50 F. Mugele, A. Klingner, J. Buehrle, D. Steinhauser and

S. Herminghaus, J. Phys.: Condens. Matter, 2005, 17, S559S576.

51 H. J. Moon, S. K. Cho, R. L. Garrell and C.-J. Kim, J. Appl. Phys.,2002, 92, 40804087.

52 R. B. Fair, Microfluid. Nanofluid., 2007, 3, 245281.53 J. Gong, Ph.D. Dissertation, Mechanical and Aerospace Engineering

Department, University of California, Los Angeles (UCLA), USA,2007, pp. 7074.

54 A. Docoslis, N. Kalogerakis and L. A. Behie, Cytotechnology, 1999,30, 133142.

55 C.-T. Ho, R.-Z. Lin, W.-Y. Chang, H.-Y. Chang and C.-H. Liu, LabChip, 2006, 6, 724734.

56 A. Jamshidi, P. J. Pauzauskie, P. J. Schuck, A. T. Ohta, P. Y. Chiou,J. Chou, P. D. Yang and M. C. Wu, Nat. Photon., 2008, 2, 8589.


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